La Crosse virus (LACV), a NIAID Category B priority pathogen, is a common cause of pediatric encephalitis and aseptic meningitis in areas of the Midwestern United States where its principal mosquito vector, Ochlerotatus (formerly Aedes) trisariatus, resides. The structural components of the LACV genome (L, M, and S) have essential, well- defined roles in virus pathogenesis. Previous studies from our laboratory using wild-type LACV and TAHV 181/57, a highly neurovirulent strain with low neuroinvasiveness, have mapped the neuroinvasive phenotype to the M segment, which encodes Gn, Gc, and a non-structural protein, NSm. More recently, using recombinant glycoproteins we demonstrated that the region corresponding to the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating fusion and cell entry. Further computational analysis identified structural similarities between LACV Gc amino acid region 970-1350 and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Collectively, these studies suggested that the LACV Gc, like the alphavirus E1 and the flavivirus E, functions as a type II fusion protein. Within Gc there is a 22 amino acid hydrophobic segment, 1066-1087, that is predicted to correlate structurally with a hydrophobic domain of SFV and Sindbis virus E1. The short sequence is highly conserved within the family Bunyaviridae and features several conserved cysteine residues, as do other type II proteins, such as SFV E1. Based on these features, and in our mutagenesis studies, our working hypothesis is that the LACV Gc (1066-1087) functions as its fusion peptide. In the first specific aim, we will extend these studies by analyzing fusion in mosquito cells, and by identifying peptides and antibodies that further associate this region with fusion and entry. In the second specific aim, we will use a newly developed reverse genetics system to construct LACV mutants incorporating the knowledge gained from the studies on the isolated glycoproteins. These viruses will then allow us to extend our in vitro findings to a mouse model of LACV encephalitis previously developed by our group (third specific aim). Importantly, as this hydrophobic region is highly conserved among the Bunyaviridae, this proposal will also elucidate mechanisms of virus fusion and entry among other emerging bunyaviruses including the NIAID Category A and C pathogens CCHFV and RVFV and will have significant implications for anti-viral therapy. PUBLIC HEALTH RELEVANCE La Crosse Virus is a common cause of pediatric encephalitis and aseptic meningitis in the Midwestern United States where it principal mosquito vector, Ochlerotatus triseriatus resides. We have identified the fusion peptide for the La Crosse virus glycoprotein Gc. The studies outlined in this proposal will define mechanisms of fusion and entry for La Crosse Virus in both the mammalian and insect host, determine the role of the newly identified fusion domain in the neuropathogenesis of LACV encephalitis, and develop anti-viral therapies (fusion peptide inhibitors and attenuated virus vaccines).